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of carbopol 934 and microcrystalline cellulose. Drug Develop Ind Pharm by ionic cross-linking technique: Morphology and release characteristics. 2001;27:381-91. Indian J Pharm Sci 2008;70:77-84. 17. Chowdary KPR, Srinivasa Rao Y. Design and in vitro and in vivo evaluation of mucoadhesive microcapsules of glipizide for oral controlled release: A Accepted 2 January 2010 technical note. AAPS PharmSciTech 2003;4:1-6. Revised 10 September 2009 18. Srinivasrao B, Ramanamurthy KV. Rifampicin hydrophilic matrix tablets: Received 4 October 2007 Design and studies. Int J Pharm Excip 2001;3:19-23. Indian J. Pharm. Sci., 2010, 72 (1): 101-105 19. Das MK, Senapati PC. Furosemide-loaded alginate microspheres prepared

Antioxidant Activity in the Extracts of Two Edible Aroids

P. MANDAL, T. K. MISRA1 AND I. D. SINGH1* Department of Botany, University of North Bengal, Darjeeling-734013, India, 1Institute of Plantation Science and Management, University of North Bengal, Darjeeling-734 013, India

Mandal et al.: Antioxidant edible aroids

Two neglected species of Araceae, Alocasia macrorhiza (Linn.) G. Don and Alocasia fornicata (Roxb.) Schott are important as food and ethno medicine in Asia and Africa. Their bioeffi cacy is documented in the Ayurveda. The solvent extracts of different edible parts of these two species like rhizomes, leaves, roots and stolons were screened for in vitro antioxidant properties using standard procedures. The successive extracts in hexane, benzene,

toluene, chloroform, diethyl ether, ethyl acetate and water fraction exhibited IC50 values in the following order, roots>rhizome>leaves for Alocasia macrorhiza and leaves>stolon for Alocasia fornicate, respectively in 2,2-diphenyl- 1-picryl hydrazyl antioxidant inhibition assay. Maximum antioxidant activity was observed in diethyl ether extracts

for both species. The IC50 values were comparable with those of quercetine and ascorbic acid as standards. These results suggest that the two aroid species have antioxidant activity in their edible parts and should be extracted using diethyl ether solvent.

Key words: Alocasia fornicata (Roxb.) Schott, Alocasia macrorhiza (Linn.) G. Don, Antioxidants, Edible aroids, Extraction, free radical scavenging

The two wild aroid perennial species A. macrorhiza It is also known as poor mans’ food primarily due and A. fornicata belong to the family Araceae, are to their richness in Starch[1]. Other phyto-constituents naturally grown in marshy land of tropical areas in are triglochinin and calcium oxalate[2]. The antioxidant India, Bangladesh, China, and South Africa. They properties of the edible parts of different plants are are also cultivated in India and Bangladesh. Typical already established[3-5]. It is widely accepted that characteristics of A. macrorhiza are 4-6 ft tall in variety of disorders such as heart disease, chronic height, light green petiole, triangular shaped wide renal failure, diabetes, cancer, immunue dysfunction, leaves and vertically geotropic rhizome with whitish ageing and life style related diseases are closely roots. The characteristics of A. fornicata are 2-3 ft tall related to the oxidation-reduction reaction in living in height, slightly pinkish colored petiole, triangular organisms[6-8]. Free radicals or reactive oxygen species shaped wide leaves and horizontally growing stolon. (ROS) are formed in vivo from different biochemical These species are commonly known as Mana kachu reactions and also from the respiratory chain as a and Shola kachu (figs. 1 and 2) and are widely result of occasional leakage from metabolic circuits. used in Ayurveda medicines in India since time These free radicals are the main sources of lipid immemorial. peroxidation[9]. Free radicals induced oxidative stress has been implicated in the pathogenesis of wide Almost all parts of these plants are used as a food. variety of clinical disorders, resulting usually from defi ciency of natural antioxidative defense[10]. *Address for correspondence E-mail: [email protected] There are growing industrial interests towards

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Fig. 1: Giant taro, Alocasia macrorhiza (linn.) G. Don. Fig. 2: Shola kachu, Aalocasia fornicata (roxb.) Schott. the production of herbal antioxidants which can at 40o and dissolved in 20 ml of methanol: water:: be supplemented in medicine, cosmetics and 4:1. Solutions of quercetine and ascorbic acid (15 µg/ nutraceuticals in modern World. This research is ml each) used as standard for in vitro studies were concerned with the demands for antioxidants in the prepared in the same solvents. developed World and also for those living below the poverty line in India, Africa and Bangladesh The antioxidant activities of the plant extract who frequently use these aroids as staple food. were determined on the basis of the free radicals Therefore, a study was under taken to screen different scavenging effect of stable DPPH free radical[11]. solvent extracts of commonly used edible parts of According to principle of the method, in the presence A. macrorhiza and A. fornicata to study the in vitro of an antioxidant, the purple colour intensity of antioxidant activities using standard procedures of DPPH solution decays and the change of absorbance DPPH based free radical scavenging activity. are followed spectrophotometrically at 517 nm. 2 ml of DPPH was added to 200 µl of different solvent The whole edible parts of A. macrorhiza (rhizome, extracts solution at different concentrations. After roots, leaves) and A. fornicata (stolon and leaves) incubation at 37o for 30 min, the absorbance of each were collected from surrounding local areas of solution was determined, the corresponding blank Phansideoa Block, Darjeeling, West Bengal, India in readings were also taken and remaining DPPH was November 2006. Plant species were authenticated. The calculated. The scavenging activity was expressed as collected plant sample were cleaned, different parts IC50 (μg/ml). IC50 value is the concentration of sample were separated and dried in the shade, then powdered required to scavenge 50% DPPH free radical. IC50 to 40 mesh and stored in an air tight container at values were calculated by applying suitable regression room temperature. Chemicals used in the studies analysis from the mean inhibitory values of DPPH were 2,2-diphenyl-1-picrylhydrazyl (DPPH), hexane, radical. All analyses were done in quadruplicate. benzene, toluene, chloroform, diethyl ether, ethyl acetate, quercetine, ascorbic acid and double distilled The successive extracts of edible parts of these plant water. All chemicals used for the experiments were of species exhibited antioxidant activity in the DPPH analytical grade. free radical assay as evidenced by the low IC 50 values (Tables 1 and 2). These values were found Different air dried ground parts (400 mg each) to be less than those obtained for the reference of these plants were soaked in 20 ml of different standards; quercetine and ascorbic acid. solvents successively [hexane, benzene, toluene, chloroform, diethyl ether, ethyl acetate and distilled Out of 7 extracts of A. macrorhiza (rhizome, roots water in order of increasing polarity] at room and leaves) and two standards tested for antioxidant temperature over night. The extracts were filtered, activity using DPPH method; the diethyl ether and the residues were macerated for two more days extract of roots and rhizome showed the maximum with the same solvent. After filtration, the solvents antioxidant activity with IC50 value 34.51±2.72 and were evaporated to dryness under reduced pressure 48.01±6.68 µg/ml, respectively. The diethyl ether

106 Indian Journal of Pharmaceutical Sciences January - February 2010 www.ijpsonline.com

TABLE 1: ANTIOXIDANT ACTIVITY OF DIFFERENT solvents like water are unsuitable for extracting EDIBLE PARTS OF ALOCASIA MACRORHIZA (LINN.) bioactive antioxidants from rhizome, roots and stolon G. DON of aroids. But in case of leaves, sufficient level of Solvent IC values±SD* (µg/ml) 50 antioxidants was found in aqueous extracts which extracts Rhizome Leaves Roots are highly comparable with reference standards. This Hexane 192.78±12.24 245.17±16.89 430.68±55.09 Benzene 829.12±52.66 128.07±8.82 127.72±16.34 information may be useful for preparing aroid based Toluene 334.69±21.26 145.44±10.02 685.90±87.74 nutraceuticals. Chloroform 688.77±95.76 225.86±15.55 112.35±8.86 Diethyl ether 48.01± 6.68 103.39±7.12 34.51±2.72 Ethyl acetate 221.14±30.75 128.07±8.82 265.56±13.58 This experiment has proved interestingly antioxidant Aqueous 560.87±17.88 58.37±6.21 164.61±8.42 activity of successive extracts of both species. They *Average of 9 determinations. Reference standard: quercetine (53.60±1.79) are widely used as food and Ayurvedic formulations. and ascorbic acid (78.17±4.05). All data were analyzed using SSP version 2.5 (2005). Certain plant species show antioxidant activity because of presence of polyphenolic constituents. TABLE 2: ANTIOXIDANT ACTIVITY OF DIFFERENT Flavonoids are a broad class of lower molecular EDIBLE PARTS ALOCASIA FORNICATA (ROXB.) SCHOTT weight, secondary metabolites extraordinarily distributed in plants. Hence, the benefi cial effects of Solvent extracts IC50 values±SD* ( µg/ml) Leaves Stolon A. macrorhiza and A. fornicata may be attributed to Hexane 69.35±3.50 128.73±29.49 their antioxidant and chelating ability of polyphenolic Benzene 62.66±3.16 191.15±9.44 constituents[14]. Toluene 400.09±20.17 180.23±41.29 Chloroform 248.85±56.14 485.22±69.15 Diethyl ether 31.11±7.02 41.23±9.44 The present investigation has indicated the presence Ethyl acetate 179.34±15.63 567.06±89.17 Aqueous 80.70±7.03 236.74±37.23 of good antioxidant activity in A. macrorhiza and A. *Average of 9 determinations. Reference standard: quercetine (53.60±1.79) fornicata. Further studies would have been required and ascorbic acid (78.17±4.05). All data were analyzed using SSP version for the isolation and purifi cation of drug to identify 2.5 (2005). the active principals which may be beneficial in a and aqueous extracts of leaves also exhibited good number of free radical mediated disorder process. It is well known that calcium oxalate is the most quantum of antioxidant activity with lesser IC50 values of 103.39±7.12 and 58.37±6.21 µg/ml, respectively. important insoluble mineral found in the edible taro However, hexane, benzene, toluene, chloroform, (Alocasia sp.). We have observed abundant spindle, and ethyl acetate extracts of rhizome and roots have star shaped crystals (fi g. 3) of calcium oxalate, which was accumulated predominantly in the cortex tissues lower antioxidant activity with higher IC50 values as presented in Table 1. The known antioxidants ascorbic of roots of A. macrorhiza. Though existence of these types of crystals is problematic for gaining popularity acid and quercetine exhibited IC50 value of 78.17±4.05 and 53.60±1.79µg/ml, respectively. but they can play effective defensive role against chewing herbivores. Diethyl ether extract of stolon and leaves of A. fornicata showed the maximum antioxidant activity with IC50 values 31.11±7.02 and 41.23±9.44 µg/ ml, respectively. The other solvent extracts proved lower antioxidant activity with higher IC50 values than standard as presented in Table 2. Similar results were also observed during successive extraction of Majorana syriaca, where diethyl ether was proved to be a best solvent for extracting antioxidants[12]. But in most cases, ethyl acetate was reported as best extracting solvent for isolating antioxidant polyphenols[13].

It was confirmed from our experiments that highly Fig. 3: Calcium oxalate crystals in roots of giant taro Alocasia hydrophobic solvents like hexane or typically polar macrorhiza (linn.) G. Don.

January - February 2010 Indian Journal of Pharmaceutical Sciences 107 www.ijpsonline.com ACKNOWLEDGEMENTS 7. Young IS, Woodside J. Antioxidants in health and disease. J Clin Pathol 2001;54:176-86. 8. Heineck JW. Oxidative stress-New approaches to diagnosis and We thank Dr. T. K. Paul, Sr. Scientist, Botanical prognosis in the atherosclerosis. Am J Cardiol Am 2003;91:12-6. Survey of India, Kolkata for identification and 9. Cheesman KH, Slater TF. Free radicals in medicine. Br Med Bull authentication of two aroids species used in this 1993;49:475-724. 10. Raj KJ, Shalini K. Flavonoids: A review of biological activities. Indian experiment. Drugs 1999;36:668-76. 11. Dhalwal K, Deshpande YS, Purohit AP, Kadam SS. Evaluation of the antioxidant activity of Sida cordifolia, J Pharm Biol 2005;43:754-61. REFERENCES 12. Al-Bandak G, Oreopoulou V. Antioxidant properties and composition of Majorana syriaca extracts. Eur J Lipid Sci Technol 2007;109:247-55. 1. Bhattacharya SK. Chiranjibi banoishidhi. Vol. 3. Kolkata: Ananda 13. Badami S, Prakash O, Dongre SH, Suresh B. In vitro antioxidant Publishers; 2005. p. 133-40. properties of Solanum pseudocapsicum leaf extracts. Indian J Pharmacol 2. Horborne JB. Secondary plant products. In: Bell EA, Charlwood BV, 2005;37:251-52. editors. Plant Phenolics: encyclopedia of plant physiology. Vol. 8. New 14. Wiseman SA, Balentine DA, Frie B. Antioxident in tea. Crit Rev Food York: Springer-Verlag; 1980. p. 329-402. Sci 1997;37:705-18. 3. Mabe K, Yamada M, Oguni I, Takahashi T. In-vitro and in-vivo activities of tea catechins against H. pylori. Am Soc Microbiol 1999;3:1788-91. 4. Hitchen CH, Gabalawy HS. Oxidation in Rheumatoid arthritis. Arthritis Accepted 3 January 2010 Res Ther 2004;6:265-78. Revised 11 September 2009 5. Pajohk F, Riedisser A, Henke M, McBride WA, Fiebich B. The effects Received 11 July 2007 of Tea extracts on proinfl ammatory signaling. BMC Med 2006;4:28-32. 6. Halliwell B. The antioxidant paradox. Lancet 2000;355:1179-80. Indian J. Pharm. Sci., 2010, 72 (1): 105-108

RP-HPLC Method for the Determination of Losartan Potassium and Ramipril in Combined Dosage Form

K. SRINIVASA RAO* AND K. SRINIVAS Roland Institute of Pharmaceutical Sciences, Berhampur-760 010, Orissa, India.

Rao and Srinivas: RP-HPLC Determination of Losartan Potassium and Ramipril

A simple, specifi c and accurate reverse phase liquid chromatographic method was developed for the simultaneous determination of losartan potassium and ramipril in table dosage forms. A hypersil ODS C18, 4.6×250 mm, 5 µm column in isocratic mode, with mobile phase acetonitrile:methanol:10 mM tetra butyl ammonium hydrogen sulphate in water in the ratio of 30:30:40% v/v/v was used. The fl ow rate was 1.0 ml/min and effl uent was monitored at 210 nm. The retention times of losartan potassium and ramipril were 4.7 and 3.3 min, respectively. The linearity range for losartan potassium and ramipril were in the range of 0.04-100 µg/ml and 0.2-300 µg/ml, respectively. The proposed method was also validated and successfully applied to the estimation of losartan potassium and ramipril in combined tablet formulations.

Key words: Losartan potassium, ramipril, validation, RP-HPLC

Losartan potassium (LRT) is chemically 2-butyl-4- carboxy-3-phenylpropyl]alanyl]octahydrocyclopenta[b] chloro-1-[p-(o-1H-tetrazol-5ylphenyl)benzyl]imidazole- pyrrole-2-carboxylic acid-1-ethyl ester. It is an 5-methanol monopotassium salt. LRT is the fi rst of a antihypertensive agent. Ramiprilat, the diacid unique class of oral antihypertensive agents referred metabolite of ramipril, is a non-sulfhydryl angiotensin to as angiotensin II receptor antagonists[1]. Ramipril converting enzyme inhibitor[2-3]. RAM is converted to (RAM) is chemically (2S,3aS,6aS)-1[(S)-N-[(S)-1- ramiprilat by hepatic cleavage of the ester group.

*Address for correspondence A literature survey regarding quantitative analysis E-mail: [email protected] of these drugs revealed that a few methods[4-13]

108 Indian Journal of Pharmaceutical Sciences January - February 2010